Therefore, inhibition of LDHA expression can also reduce the amount of pyruvate that is oxidized in the citric acid cycle and mitochondrial respiration. Lactate is known to fuel mitochondrial respiration in cancer cells and ECs 8 , We confirm here that inhibition of LDHA expression decreases basal respiration levels and glucose uptake levels, but does not affect total ATP levels and glycolytic activity.
Additionally, inhibition of expression of the glycolytic gene PFKFB3 partially reduces glycolysis, but increases mitochondrial respiration.
Collectively, our outcomes support the findings that the glycolytic end-product lactate is a substrate that can fuel mitochondrial respiration to generate ATP and that ECs, similarly to cancer cells, are able to switch to mitochondrial respiration when glycolysis is inhibited 23 , 67 , In conclusion, our detailed studies show a complex role of the different metabolic pathways in the specific functions of tip cells and non-tip cells and in the regulation of angiogenesis.
Glycolysis regulates initial tip cell formation, and lactate is essential for tip cells to maintain their phenotype. On the other hand, tip cells are more dependent on glucose consumption for their mitochondrial functioning as compared to non-tip cells, whereas glycolysis as well as mitochondrial respiration are essential for the proliferation of non-tip cells.
Finally, we demonstrate that the metabolic flexibility of ECs allows the switch between metabolic pathways, e. Together, our findings confirm previous findings on the metabolic flexibility of tip cells and non-tip cells and verify that ECs can adapt to the rapidly changing microenvironment during sprouting angiogenesis 19 , and show that glycolysis as well as mitochondrial respiration are essential for angiogenesis in vitro as well as in vivo.
These important features of ECs may have considerable significance in understanding the complex functioning of metabolism in endothelial tip cells and non-tip cells. The percentage of tip cells in cell cultures was determined as described earlier The fluorescein isothiocyanate FITC channel was used to detect autofluorescence.
Non-stained and non-treated cells were used as negative controls. A schematic overview of the relevant genes is shown in Fig.
All experiments were performed in triplicate. Louis, MO, USA , a glucose analog that inhibits the initial step of glycolysis by its interaction with hexokinase Fig. To determine glucose uptake levels, cells were incubated with a fluorescent D-glucose analog 2-[N- 7-nitobenzoxa-1,3-diazolyl -amino]deoxy-D glucose 2-NBDG; Fig. OCR, a measure of oxygen utilization of cells, is an important indicator of mitochondrial function Extracellular acidification rate ECAR is a measure of lactic acid levels, formed during the conversion of glucose to lactate during glycolysis The following inhibitors were injected: oligomycin A 1.
This allowed for calculation of OCR-linked ATP production, maximal respiration capacity and spare respiratory capacity. Basal respiration was measured prior to addition of oligomycin A. This allowed for calculation of glycolysis rate, glycolytic capacity, and glycolytic reserve. Basal ECAR was measured prior to addition of glucose. After blocking with 2. Total sprout length and the number of sprouts were quantified of at least 10 spheroids for every condition.
All analyses were carried out in a blinded fashion. Quantification was carried out using Neuron-J plug-inn package for Image-J software The in ovo CAM assay was performed as described earlier 73 , On embryo developmental day EDD 3, a small hole was prepared in the eggshell and covered with parafilm Pechinery, Menasha, WI, USA to prevent dehydration and possible infections and eggs were returned to the incubator. The latter was analyzed and quantified manually by two independent observers and carried out in a blinded fashion.
All experiments were performed in HUVECs of at least 3 donors and were performed in duplicate or triplicate. To correct for differences between donors, factor correction, as described previously 77 , was used for flow cytometry data, spheroid data, and Seahorse flux data.
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Learning Objectives The theoretical maximum yield of ATP for the oxidation of one molecule of glucose during aerobic respiration is In terms of substrate-level phosphorylation, oxidative phosphorylation, and the component pathways involved, briefly explain how this number is obtained. Contributors and Attributions Dr.
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